Nitroaromatic and nitramine compounds comprise a class of pollutants known to have both toxic and carcinogenic properties. Nitroaromatic and nitramine pollutants are frequently generated in the production of explosives, such as TNT (2,4,6-trinotrotoluene), RDX (Royal demolition explosive; hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (High melting point explosive; octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) (see FIG. 1). In particular, due to their low solubility, large quantities of water used in the production of explosives tend to become contaminated during their production, leading to wastewater disposal problems.
First synthesized in 1863, TNT was used in the dye industry before becoming in the 20th century the main conventional explosive used worldwide. However, because of a higher stability and detonation power, nitramines HMX and RDX are at the present time the most widespread conventional explosives. Manufacture of nitro-substituted explosives, testing and firing ranges, and destruction of ammunition stocks have generated toxic wastes leading to large-scale contamination of soils and groundwater (44). Seven nitro-substituted explosives, including TNT and RDX, have been listed as priority pollutants by the U.S. Environmental Protection Agency (EPA) (25). RDX, formerly used as a rat poison, is in addition considered as possible carcinogen by the EPA (2, 28). HMX has been listed as an EPA contaminant of concern (48). A lifetime health advisory of 2 μL−1 of TNT in drinking water and a water-quality limit of 105 μg L−1 of RDX have been recommended (7, 37). Physicochemical properties, biodegradation, and toxicity of nitro-substituted explosives have been extensively reviewed in the literature (12, 18, 38, 43, 45, 53).
The toxicity of TNT has been reported since the First World War among English ammunition workers. From laboratory studies TNT, RDX, and HMX have been found to be toxic for most classes of organisms, including bacteria (46, 57), algae (46, 57), plants (35), earthworms (36), aquatic invertebrates (46, 48), animals (22, 42), mammals (28, 48), and humans (3, 22).
Traditional treatments of toxic ammunition wastes (i.e. open burning/open detonation (OB/OD), adsorption onto activated carbon, photooxidation (UV/O3), etc.) are costly, damaging for the environment, and in most cases practically infeasible.
Biotransformation of energetic pollutants TNT, RDX, and HMX have been reported for different classes of organisms, including bacteria, fingi, and plants (12, 18, 29, 33, 38, 41, 53). Metabolism of TNT typically involves a sequential reduction of the nitro groups to form toxic aromatic amino derivatives, which are poorly further transformed (29, 33). Except with white-rot fungi, that secrete powerful ligninolytic peroxidases (8, 54), no significant mineralization has been detected in biological systems (29). In contrast to TNT, whose limiting degradation step is the aromatic ring fission, as soon as nitramines RDX and HMX undergo a change of the molecular structure, the ring collapses to generate small aliphatic metabolites (17, 53). While other decomposition mechanisms have been reported (i.e. concerted decomposition, bimolecular elimination, or hydrolysis (17)), biotransformation of RDX and HMX frequently involves an initial reduction of the nitro groups to form nitroso and hydroxylamino derivatives (31). The latter decompose to unstable aliphatic nitramines, eventually converted into N2O and CO2 (17,18). Due to different conformations, HMX (crown-type) is chemically more stable and therefore less amenable to biodegradation than RDX (chair-type) (17).
Bacteria of the genus Methylobacterium are strictly aerobic, facultative methylotrophic, Gram-negative, rod-shaped bacteria that are able to grow on one-carbon compounds, i.e. methanol or methyamine (13, 30, 51). Members of the genus Methylobacterium, which belongs to the α-2 subclass of Proteobacteria, are distributed in a wide diversity of natural and human-made habitats, including soils, air, dust, fresh water, aquatic sediments, marine environments, water supplies, bathrooms, and masonry (19, 51). Some species have been described as opportunistic human pathogens (51). In addition, methylotrophic bacteria colonize the roots and the leaves of terrestrial and aquatic plant species (21, 34, 51). These bacteria are often red to pink due to the presence of carotenoids and referred as pink-pigmented facultative methylotrophs (PPFMs). Methylobacterium bacteria are highly resistant to dehydration, freezing, chlorination, UV light, ionizing radiations, and elevated temperatures (51).